Autocamtide 2, amide

The Ca(2+)-dependent facilitation (CDF) of L-type Ca(2+) channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca(2+)/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca(2+) currents (I(Ca,L)) by H(2)O(2) and whether Ca(2+) is required in this process. Using patch clamp, I(Ca)(,L) was measured in rat ventricular myocytes. H(2)O(2) induced an increase in I(Ca,L) amplitude and slowed inactivation of I(Ca)(,L). This oxidation-dependent facilitation (ODF) of I(Ca)(,L) was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca(2+) with Ba(2+) or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca(2+) stores using caffeine and thapsigargin. Alkaline phosphatase, beta-iminoadenosine 5'-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca(2+) channels is mediated by oxidation-dependent CaMKII activation, in which local Ca(2+) increases induced by SR Ca(2+) release is required.

Background: Digitalis-induced Na(+) accumulation results in an increase in Ca(2+)(i) via the Na(+)/Ca(2+) exchanger, leading to enhanced sarcoplasmic reticulum (SR) Ca(2+) load, responsible for the positive inotropic and toxic arrhythmogenic effects of glycosides. A digitalis-induced increase in Ca(2+)(i) could also activate calcium-calmodulin kinase II (CaMKII), which has been shown to have proarrhythmic effects. Here, we investigate whether CaMKII underlies digitalis-induced arrhythmias and the subcellular mechanisms involved. Methods and results: In paced rat ventricular myocytes (0.5 Hz), 50 μmol/L ouabain increased contraction amplitude by 160 ± 5%. In the absence of electric stimulation, ouabain promoted spontaneous contractile activity and Ca(2+) waves. Ouabain activated CaMKII (p-CaMKII), which phosphorylated its downstream targets, phospholamban (PLN) (Thr17) and ryanodine receptor (RyR) (Ser2814). Ouabain-induced spontaneous activity was prevented by inhibiting CaMKII with 2.5 μmol/L KN93 but not by 2.5 μmol/L of the inactive analog, KN92. Similar results were obtained using the CaMKII inhibitor, autocamtide-2 related inhibitory peptide (AIP) (1 to 2.5 μmol/L), and in myocytes from transgenic mice expressing SR-targeted AIP. Consistently, CaMKII overexpression exacerbated ouabain-induced spontaneous contractile activity. Ouabain was associated with an increase in SR Ca(2+) content and Ca(2+) spark frequency, indicative of enhanced SR Ca(2+) leak. KN93 suppressed the ouabain-induced increase in Ca(2+) spark frequency without affecting SR Ca(2+) content. Similar results were obtained with digoxin. In vivo, ouabain-induced arrhythmias were prevented by KN93 and absent in SR-AIP mice. Conclusions: These results show for the first time that CaMKII mediates ouabain-induced arrhythmic/toxic effects. We suggest that CaMKII-dependent phosphorylation of the RyR, resulting in Ca(2+) leak from the SR, is the underlying mechanism involved.

Introduction: Calcium/Calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase that phosphorylates and regulates activity of many substrates in various tissues. Traditional CaMKII activity assays rely on incorporation of radioactivity onto a CaMKII substrate by utilizing γ-32P ATP, which has a short half-life and can pose health risks to the researchers. Methods: An 8-minute HPLC-MS method was developed to measure a CaMKII-specific peptide substrate autocamtide-2 (AC-2) and its phosphorylated form, phosphoautocamtide-2 (PAC-2). Degradation of AC-2 and PAC-2 in solutions and how to stabilize them were studied. The method was validated according to FDA guidelines for bioassays, and applied to determine CaMKII activity in a C2C12 cell lysate and IC50 of KN-93, a known CaMKII inhibitor. Results: Simple acidification with formic acid prevented AC-2 and PAC-2 from undergoing rapid degradation in the CaMKII assay mixture and in diluted water solutions. LLOQ of the HPLC-MS method was 0.26 μM and 0.12 μM for quantification of AC-2 and PAC-2, respectively. Precision was within 15% and accuracy was within 100 ± 15%. Using the developed method, IC50 of KN-93 was measured to be 399 ± 66 nM, which was compatible to reported values. Conclusions: A validated HPLC-MS method provides precise and accurate determination of AC-2 and PAC-2. This method enabled enzyme activity assay and inhibitor IC50 determination for CaMKII without radioactive labelled reagents.